Bio-macromolecules, such as proteins, nucleic acids and polysaccharides, may often partially occur in the form of aggregates, or multimers, such as dimers, trimers or higher oligomers or aggregates. In, for example, recombinant DNA technology, where desired polypeptides or proteins are produced in host organisms and isolated from cell extracts under conditions and in concentrations quite different from those in their natural environment, the conditions may favour the formation of such aggregates through intermolecular disulphide linkages or other covalent bonds, or through non-covalent interactions.
The presence of such aggregates of a target macromolecule are many times undesired. Protein aggregation is thus a common issue encountered during bioprocess development and manufacturing of biotherapeutics. Since the multimeric forms of the macromolecule may have lower or lack the biologic activity, or even cause undesired side-effects, it is essential for therapeutic safety that the therapeutic protein is in a monomeric state and that there are no aggregates of molecules present. It is consequently of importance that the amount of aggregates produced during cell culturing and the purification process can be controlled by the implementation of appropriate measures.
The analysis of aggregates in this context is today mainly performed by size-exclusion chromatography, sometimes coupled with light scattering detection. This method is relatively slow and complex to perform and therefore not readily amenable for screening purposes with demand for larger sample volumes.
WO 2004/040317 A1 discloses a sensor device and method for determining the extent of aggregation of a protein, such as beta-amyloid, in fluid, e.g. a bodily fluid. The sensor device has a sensing layer provided with a binding partner to the protein and is sensitive to changes in the localised environment of the sensing layer caused by the introduction of the fluid. The response is typically related to changes in volume and mass from which changes in molecular density are calculated. The extent of aggregation of the protein is directly related to a change in molecular density, lowly aggregated protein giving rise to a significant increase in molecular density, and specific binding of highly aggregated protein giving rise to a less significant increase or a decrease in molecular density. The sensor device is particularly an interferometric type waveguide structure (interrogated by electromagnetic radiation in TE mode and TM mode), but also a piezoelectric sensing system, or a surface plasmon resonance device in combination with ellipsometry are suggested as sensor devices.
It is an object of the present invention to provide an improved and simplified method for assessing the content of aggregates in a fluid containing the macromolecule, especially an antibody preparation.